The Cdc25 phosphatases positively regulate the cell cycle by activating Cdks. In mammals, three family members, denoted Cdc25A, Cdc25B, and Cdc25C, exist. It is unclear why higher eukaryotic organisms encode three Cdc25s while yeast cells survive with one. Mice that can be deleted for Cdc25A were generated. In addition, mice lacking Cdc25B, Cdc25C or both and that can also be conditionally deleted for Cdc25A were generated. These mice will be used to determine the contributions made by Cdc25 family members to adult cell cycles. Another goal of this proposal is to utilize high throughput and molecular imaging strategies to identify novel regulators of Cdc25A. Cdc25A functions throughout the cell cycle to regulate cell cycle transitions and Cdc25A is overproduced in several cancers. Cdc25A activity, stability and interactions with other proteins are regulated by reversible phosphorylation. Thus, identifying the protein kinases that regulate Cdc25A will provide insight into how Cdc25A is regulated under normal and stressed conditions and what pathways leading to Cdc25A overproduction are derailed in human cancers. Finally, experiments are proposed to study a novel regulatory pathway involving ATR, Chk1 and PP2A and to investigate interactions between Chk1 and Cdc25A in mice. In addition to advancing our understanding of basic cell cycle principles, these studies have clinical impact as well. For example, Cdc25A and Cdc25B are overproduced in many human cancers and efforts are underway to develop Cdc25 inhibitors that can be used to treat human cancers. The cytotoxicity of Cdc25 inhibition needs to be assessed before Cdc25 inhibitors can proceed to the clinic. The gene knockout studies proposed in this grant will assess effects of loss of Cdc25 family members in adult mice and may predict how patients will respond to global versus individual Cdc25 inhibition. Another strategy that is currently being used to treat cancer patients is to combine DNA damaging agents with drugs that induce Cdc25A accumulation. In preclinical models, this strategy induces checkpoint bypass and preferential killing of p53-deficient cells. The Chk1 inhibitor UCN-01 is being tested with DNA damaging agents in Phase I and II clinical trials. Thus, the novel kinases and/or regulators of the PP2A/Chk1 pathway identified in the course of our studies are potential druggable targets. Inhibitors that target components of these pathways could substitute for Chk1 inhibitors in the combination therapy described above. The hope is that these inhibitors may induce less genome instability than Chk1 inhibitors. PUBLIC HEALTH RELEVANCE: Experiments are proposed to fully dissect the molecular underpinnings of the PP2A/Chk1/Cdc25A regulatory pathway and to functionally characterize the Cdc25 phosphatases in mice. The information gained from the proposed studies is expected not only to enhance our understanding of basic principles of cell cycle control in mammals but is also expected to directly impact future therapeutic strategies for cancer treatment.